Human papilloma virus genes and their use in gene therapy

ABSTRACT

A process of immortalizing cells with isolated HPV-16, 18, 31, 33 or 35 E6 and E7 genes or the E7 gene alone to produce non-tumorigenic immortalized cell lines which retain the differentiated phenotypic characteristics of the parent cells.

This application is a divisional of application Ser. No. 07/874,397,filed Apr. 27, 1992, now U.S. Pat. No. 5,376,542.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the immortalizing E6 and E7 genesof human papilloma viruses, such as virus types 16, 18, 31, 33 and 35and transfection of these genes into target cells. In vivo expression ofE6/E7 genes immortalizes the target cell which retains its phenotypiccharacteristics.

2. Discussion of the Background

More than 50 different types of human papillomariruses (HPVs) have nowbeen isolated from a variety of squamous epithelial lesions, andapproximately 18 of them have been associated with anogenital tractlesions. Some of these, such as HPV type 6 (HPV-5) and HPV-11, aregenerally associated with benign proliferative lesions, includingcondyloma acuminata, which only infrequently progress to cancers.Others, such as HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35, areassociated with genital tract lesions, which are at risk for malignantprogression, and with genital tract cancers (1).

HPV-16 or HPV-18 DNA has been found integrated in a high percentage ofcervical carcinomas and in cell lines derived from these cancers (2, 3,4). This is in contrast with the premalignant dysplastic lesionsassociated with HPV-16 and HPV-18, in which the viral DNA is usuallyfound in an extrachromosomal state (5). In several cases in which thenumber of integrated viral genomes was low enough to permit a detailedanalysis, the integration pattern revealed remarkable specificity withrespect to the circular viral genome. Integration occurs in the E1-E2region (6, 7, 8), disrupting the E2 viral transcriptional regulatorycircuitry. The E2 open reading frame (ORF), as originally demonstratedwith the bovine papillomavirus type 1, encodes both positive- andnegative-acting transcriptional regulatory factors (9, 10). For HPV-16and HPV-18, E2 appears to act principally as a repressor of the promoterfrom which the E6 and E7 genes are transcribed (11, 12). The HPV genomesin cervical carcinomas and in derived cell lines are transcriptionallyactive, and the patterns of viral mRNA species are specific, withregular expression of the E6 and E7 ORRFs (6, 13, 14).

The E7 ORF of HPV-16 encodes a 21-kilodalton phosphoprotein (14), andthe E7 genes of HPV-16 and HPV-18 are sufficient for focus formation ofestablished rodent fibroblasts such as NIH 3T3 cells (15, 16, 17, 18,19). The E7 protein is functionally and structurally related to theadenovirus E1A proteins (AdE1A); it can transactivate the AdE2 promoterand can cooperate with an activated ras oncogene to transform primaryrat cells (16, 20). The amino-terminal 38 amino acids of E7 arestrikingly similar to portions of conserved domain 1 (amino acids 37 to49) and domain 2 (amino acids 116 to 137) of the AdE1A proteins (16) aswell as to portions of the large tumor antigens (T) of papovaviruses.The AdE1A, simian virus 40 (SV40) T, and HPV-16 E7 proteins formspecific complexes with the product of the retinoblastoma tumorsuppressor gene (p105-RB) (21, 22, 23), and complex formation withp105-RB is mediated through these conserved sequences for AdE1A and SV40T (21, 22) as well as for HPV-16 E7. The transforming potential of theE6 gene has been less well defined. In NIH 3T3 fibroblasts, it maycontribute to characteristics of the transformed phenotype such asanchorage independence (25) or tumorigenicity in nude mice (26). Inhuman cells, E6 appears to cooperate with the E7 oncopprotein inmediating-cellular immortalization. Recently, it has been demonstratedthat E6 binds to, and mediates the degradation of, the cellular tumorsuppressor protein p53. It has been shown recently that both the E6 andE7 RFs are necessary for the extension of the life span of human diploidfibroblasts (34). Mutation studies of the early HPV-16 genes thatdirectly participate in the in vitro transformation of primary humankeratinocytes has shown that both the full-length E6 and E7 genes arerequired for induction of keratinocyte immortalization and resistance toterminal differentiation (35). Keratinocyte transformation with HPV-18DNA requires only the HPV-18 regulatory region and the E6/E7 genes whichinduce two progressive steps in cellular transformation (36).

A quantitative keratinocyte assay has been used to demonstrate that theHPV genes can alter the response of human keratinocytes to inducers ofterminal differentiation. HPV-16 and HPV-18 DNA can immortalize humankeratinocytes in vitro. These immortalized cell lines exhibit alteredcharacteristics of cellular proliferation and differentiation but arenot tumorigenic in nude mice. They contain integrated copies of HPV DNAand express viral mRNAs and proteins (27, 28, 29, 30, 31, 32). While theabove in-vitro assays rely on the growth of keratinocytes on plasticsubstrate and can be criticized for its lack of physiologic relevance,other in vitro assays which more closely mimic the in vivo conditionsfor squamous cell growth have demonstrated similar results. Thus, whenhuman keratinocytes are grown by the collagen raft cell culturetechnique, they display an enhanced cellular differentiation andstratification which is very similar to, but not identical with, thatobserved of keratinocytes in vitro. This technique has been successfullyapplied to the study of HPV gene effects on cellular differentiation andseveral laboratories have demonstrated that the E6/E7 genes producealtered cellular differentiation reminiscent to that observed incervical dysplasia (33). Thus, all experimental data to date suggeststhat the expression of the E6/E7 genes results in a poorlydifferentiated phenotype when assayed in vitro.

(In a recent study, HPV-16 immortalized human keratinocytes weresubcutaneously injected into nude mice. Although the immortalized cellsretain the ability for differentiation after injection, the injectedcells were immunoisolated to form encapsulated cysts).

The study of normal cell growth and differentiation would be greatlyaugmented by the development of an efficient method for obtaining humanimmortalized cell lines which would retain their ability todifferentiate and respond to external regulatory signals. One criticalresearch area which would greatly benefit from such an approach would bethe study of cystic fibrosis (CF). Not only would CF cell lines permitthe analysis of the altered ion permeability properties of these cellsand their alteration by pharmacologic agents, but they would also serveas a substrate for future gene therapy experiments. In an attempt togenerate such cell lines, the SV40 large T antigen has been used toimmortalize CF cells. Unfortunately, the derived cell lines lose many oftheir differentiated properties and are inadequate for biochemical,physiological, and molecular analysis.

A need continues to exist for a means of immortalizing human cells in anon-tumorigenic manner such that the immortalized cells retain theirdifferentiated phenotypic properties. A broadly applicable means forproducing non-tumorigenic immortalized cell lines would facilitateresearch on gene products of particular cells, provide cell linescapable of producing large quantities of gene products without the needto replace senescent cell lines; provide immortalized cell lines for usein direct gene therapy applications and also provide a means ofimmortalizing cell lines containing exogenous genes or genes which havebeen subjected to site-specific mutation to correct abnormalities ingene expression.

SUMMARY OF THE INVENTION

One object of the present invention is a method of immortalizing cellsby a process in which the immortalized cells retain their differentiatedphenotypic properties and characteristics in vivo and arenon-tumorigenic.

Another object of the invention is a process for immortalizing cellswhich is widely applicable to a broad variety of different cell types.

A further object of the invention is a method of gene therapy in whichcells from a patient are removed from the patient and immortalized toproduce non-tumorigenic immortalized cells retaining the differentiatedphenotypic characteristics of the original parent cells, and thenreintroducing the immortalized cells into the patient as a therapeutictreatment.

Still a further object of the invention is a method of gene therapy inwhich cells having a defective gene are obtained from a patient. Thedefective gene can then repaired by site-specific mutation or bereplaced by an exogenous gene capable of expressing the desired geneproduct. This genetically altered host cell can then immortalized toproduce a non-tumorigenic immortalized cell line expressing the desiredgene product.

These and other objects of the invention which will become apparent fromthe following specification have been achieved by the present process ofimmortalizing cells with isolated HPV-16, 18, 31, 33 or 35 E6 and E7genes or the E7 gene alone to produce non-tumorigenic immortalized celllines which retain the differentiated phenotypic characteristics of theparent cells.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 [SEQ ID NOS. 1-5] diagrammatically shows the amplification andcloning of the LCR-E6-E7 region of HPV-16 and HPV-18 from human tumors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

HPV-16, 18, 31, 33 and 35 DNA has been shown to immortalize humankeratinocytes in vitro. However, continued growth of thesenon-tumorigenic cell lines in vitro results in altered or dysplasticcellular proliferation and differentiation characteristics. That is,keratinocytes immmortalized with HPV E6/E7 genes and grown in vitro losesome or many of the differentiated phenotypic characteristics of theparent keratinocytes. Non-tumorigenic immortalized cells are notsuitable for gene therapy applications due to the loss of differentiatedphenotypic characteristics.

Surprisingly, it has now been discovered that epithelial andnon-epithelial cells which have been immortalized with HPV-16, 18, 31,33 or 35 E6 and E7 genes or the E7 gene alone, retain the differentiatedphenotypic characteristics of the parent epithelial and non-epithelialcells when grown in vivo in the host animal after immortalization. Theloss of phenotypic characteristics which is seen when the immortalizedcells are grown in vitro is prevented when these cells are grown in vivoin the host animal. Immortalized host cells which retain the phenotypiccharacteristics of the parent cells are not immunogenic to the hostimmune system and are therefore useful for gene therapy.

By "retain the phenotypic differentiated characteristics" of parentcells used herein, is meant that the phenotypic characteristics ofstable immortalized cells are indistinguishable from parent cells, whenthe immortalized cells are grown in vivo. In vivo growth can be in anude mouse or in the host mammal or human patient. The phenotypiccharacteristics can be easily determined by conventional methods such ashistologic examination using standard strains. Where the immortalizedcells express a gene product, the cell "retains the phenotypicdifferentiated characteristics" of the parent cell when it expesses thegene product in vivo or invitro. By "stable, immortalized cells" it ismeant that the immortalized cells which are administered into the hostanimal will proceed to grow and differentiate normally and will notundergo induced terminal differentation and death subsequent to immunerejection.

Human tumors containing HPV E6/E7 genes are widely available asdescribed above. A scheme for the isolation of HPV E6/E7 genes fromhuman tumors is illustrated in FIG. 1. LCR is the HPV vital promoter. Inbrief, cellular DNA from carcinomas of the uterine cervix, vulva andpenis from patients is purified using standard techniques (Sambrook etal, Molecular Cloning: A Laboratory Manual, second edition. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989). The HPV DNA presentin each sample is typed by Southern hybridization and only those samplescontaining HPV-16 or HPV-18 DNA are further utilized.

PCR techniques for amplifying a selected DNA sequence are well known inthe art. See Erlich, H. A., PCR Technology--Principles and Applicationsfor DNA Amplification, Stockton Press, New York, N.Y. 1989. The PCRtechniques described in this reference can be readily used to amplifythe HPV E6 and E7 genes are were then inserted into a cloning vectorusing standard recombinant DNA technology.

Purified tumor DNA is amplified by polymerized chain reaction (PCR)using the designated oligonucleotides corresponding to the 5' end of theLCR promoter (defined as the terminus of the L1 OFR) and that 3' end ofthe E7 gene (immediately following the termination codon). In the caseof HPV-18, an additional oligonucleotide is used to amplify the LCR froman internal position (bp 7201). For each of the oligonucleotides (seeFIG. 1), a non-homologous 5' terminus encoding either a restriction site(HindIII or Xhol, for example) is included to facilitate theunidirectional cloning of the amplified fragment into a mutable plasmid.(Baker et al, in Recombinant Systems in Protein Expression, pages 75-86,Elsevier, Amsterdam/New York, 1990). The orientation of the amplifiedHPV LCR E6-E7 region can be verified by direct sequencing.

The plasmids constructed as described above can be evaluated in aquantitative transformation assay and compared with full-length genomesof HPV-16 and HPV-18 to verify the presence of the HPV genes in theplasmid. HPV-18 has a transformation efficiency approximately 10 foldgreater than HPV-16. The relative transforming activity of HPV-18 toHPV-16 is maintained in a subgenomic fragment containing only theLCR-E6-E7 region, indicating that other viral genes are not responsiblefor this difference in activity.

The HPV E6-E7 genes can be incorporated into any standard cloningvector. The term "vector" is well understood in the art and issynonymous with the often-used phrase "cloning vehicle". A suitablevector is a non-chromosomal double-stranded DNA comprising an intactreplicon such that the vector is replicated when placed within aunicellular organism.

Viral vectors include retroviruses, adenoviruses, herpes virus,papovirus, etc. Other suitable vectors include plasmids. Plasmids andretroviruses and preferred vectors.

In FIG. 1 [SEQ ID NOS. 1-5], pUC18 was constructed and contained thenatural HPV LCR viral promoter. This promoter is suitable for expressionof the E6-E7 genes in a wide variety of cells including both epithelialand non-epithelial cells. However, the LCR promoter is not specificallyrequired for transcription and expression of the E6-E7 genes.Optionally, one may replace the LCR promoter with other known promotersto improve the efficiency of transcription and expression in particularcells.

Many promoters optimally active in specific cell types are known in theart. For example, immortalization of the following cell types can beimproved by using the specific known promoter identified: musclecells/myosin promoter, fibroblasts/actin promoter, pancreascells/insulin promoter, bowel/lactase or sucrase, prostate cells/acidphosphatase promoter, liver cells/albumin promoter and lung cells/cysticfibrosis (CF) gene promoter.

The promoter DNA can be amplified using PCR technology whileconcurrently providing restriction sites at the 5' and 3' ends of thepromoter DNA. The amplified promoter DNA can then be inserted into acloning vehicle (for example pUC18) using conventional endonucleases andknown recombinant DNA technology. Cloning vectors containing the desiredpromoter upstream of the 5' end of the E6 or E7 genes are constructed inthis manner.

Immortalization of cells with E6-E7 genes proceeds with high efficiency.Additionally, it is possible to immortalize cells using only the HPV E7gene. Cloning vectors containing an appropriate promoter and the E7 geneare constructed using PCR technology in a manner analogous to thepreparation of vectors containing E6/E7 genes described above. Forexample, recombinant retrovirus plasmids containing HPV genes E6 and E7(Galloway et al, J. Virol. 65:473-478. 1991) are digested withexonuclease enzymes to remove the upstream E6 gene portion leaving anintact E7 gene. The E7 gene is then amplified and provided withappropriate restriction sites using PCR technology in the mannerdescribed above. The E7 gene is then inserted into an appropriatecloning vector using recombinant DNA technology after selecting andincorporating the desired promoter DNA sequence as described above. Inthis manner, a cloning vehicle containing the promoter-E7 gene isconstructed.

Cloning vectors containing HPV-16, 18, 31, 33 or 35 E6/E7 genes aretransfected into host cells using known transfection processes. Forepithelial cells, suitable transfection processes are lipofection,electroporation and retrovirus infection. For non-epithelial cells, useof calcium phosphate according to known procedure of van der Eb issufficient to achieve transfection. Retrovirus transfection is thepreferred mode of transfection for both epithelial and non-epithelialcells. Lipofection is a preferred alternative method for transfection ofepithelial cells. Electroporation according to the method of Schlegel etal (30) is a preferred second alternative for transfection oflymphocytes. Calcium phosphate transfection according to the method ofvan der Eb is a preferred second alternative for transfection ofmesenchymal cells and fibroblasts.

When transfecting cells with electroporation, the desired cells areisolated and cultured in suitable media. Electroporation is conductedusing commercially available equipment according to known methods usingoptimum voltage and current settings. The voltage and current settingscan be readily determined by one having ordinary skill in the art inconsultation with the manufacturers manual accompanying conventionalelectroporator devices. The isolated cells are electroporated with 1-20micrograms of linearized HPV or plasmid DNA containing the promoter andE6/E7 genes. Transfected cells are then replated into growth medium andanalyzed for microcolony formation after several days. The efficiency ofimmortalization following electroporation in approximately 5-20transformants/106⁶ cells electroporated.

Transfection of cells using lipofection is conducted according tostandard lipofection procedures. See Felgner et al. 1987. Proc. Natl.Acad. Sci. (U.S.A). 84:7413-7417. In general, liposome-mediated DNAtransfection is accomplished by exposing 1-20 micrograms of plasmid DNAand commercially available liposomes (Bethesda Research Laboratories) inculture medium. The transfected cells are then repeatedly passaged inculture medium and the desired clones are isolated. The efficiency ofimmortalization by lipofection in approximately 50-100/10⁶ cellslipofected.

Retrovirus infection is also accomplished using previously describedprocedures. See for example Miller et al. J. Virol. 62:4337-4345 andHalbert et al. J. Virol. 65:473-378. 1991. In general, plasmid DNA istransfected into a desired packaging cell line such as Psi-2 or othercell lines, using standard calcium phosphate precipitation. Virusesproduced from the Psi-2 cells or equivalent cells are then used toinfect an amphotropic packaging cell line, for example PA317. Virusesproduced by the amphotropic packaging cell line are used to infect thedesired host parent cells of the present invention. Immortalizationefficacy with retroviruses approximates >1000 transformants/10⁶ cellsinfected.

After transfection, the desired clones are selected by culturing inoptimal media and repeated passaging. Generally, 10-20 passages arerequired to eliminate spurious cells and obtain pure clonal cells.Optimal media are selected according to the type of parent cell which isimmortalized. For lymphocytes, RPMI media is preferred; for fibroblasts,DMEM media is preferred; and for epithelial cells, a serum-free mediumsuch as keratinocyte growth medium (KGM) or SFM (Gibco Company) ispreferred.

Selected colonies are then tested to verify the presence of HPV DNA andthe expression of E6/E7 genes. Verification is confirmed by standardSouthern hybridization techniques and immunoprecipitation to determinethe presence of expressed E6 and E7 proteins (36).

The present invention provides a means for immortalizing any cell typeto produce cells which retain the differentiated phenotypiccharacteristics of the parent cells. Epithelial cells are particularpreferred host cells for immortalization using the method of the presentinvention.

Specific epithelial cells which can be immortalized using the presentmethod include those lining the respiratory, gastrointestinal,genitourinary and nervous systems. Examples of such epithelial cellsinclude those of the oral and nasal mucosa, larynx, trachea, lung,esophagus, stomach, duodenum, jejumin, ileum, and colon. Otherepithelial cells which can be immortalized include liver, pancreas,kidney, bladder, adrenal and reproductive organ epithelial cells, suchas cells obtained from the ovary, uterus (endometrium), testis andprostate. Skin cells can also be immortalized, in particular hairfollicle cells and dermal papillae which are necessary for hair follicledevelopment.

In addition to epithelial cells, non-epithelial cells such asendothelial cells, fibroblasts, muscle cells, bone cells, cartilagecells and brain tissue cells (neurons, glial cells, etc.) can also beimmortalized using the method of the present invention. Further,hematopoietic cells such as red blood cell progenitor cells, white bloodcell progenitor cells and megakariocytes can all be immortalized. Theseimmortalized hematopoietic cells can be reintroduced into a host mammalduring gene therapy to treat blood system diseases such as aplasticanemia, leukemia and lymphomas.

Gene therapy refers to the transfer and stable insertion of new geneticinformation into cells for the therapeutic treatment of diseases ordisorders. The foreign gene is transferred into a cell that proliferatesto spread the new gene throughout the cell population. Known methods ofgene transfer include microinjection, electroporation, liposomes,chromosome transfer, and transfection techniques as well ascalcium-precipitation transfection techniques as disclosed above.

Disorders that can be treated by infusion of immortalized cells includebut are not limited to three broad categories. First are diseasesresulting from a failure or dysfunction of normal blood cell productionand maturation (i.e., aplastic anemia), as well as neoplastic, malignantdiseases in the hematopoietic organs (e.g., leukemia and lymphomas). Thesecond group of disorders comprises those of patients with broadspectrum of malignant solid tumors. The third group of diseasescomprises a number of genetic disorders which can be corrected byinfusion of immortalized cells, which prior to transplantation have beenmodified to contain an exogenous gene.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of gene therapy, in accordance with this embodiment of theinvention. The technique used should provide for the stable transfer ofthe heterologous gene sequence to the stem cell, so that theheterologous gene sequence is heritable and expressible by stem cellprogeny, and so that the necessary development and physiologicalfunctions of the recipient cells are not disrupted. Techniques which maybe used include but are not limited to chromosome transfer (e.g., cellfusion, chromosome-mediated gene transfer, micro cell-mediated genetransfer), physical methods (e.g., transfection, spheroplast fusion,microinjection, electroporation, liposome carrier), viral vectortransfer (e.g., recombinant DNA viruses, recombinant RNA viruses) etc.(described in Cline, M. J., 1985, Pharmac. Ther. 29:69-92.)

In general, for the treatment of cancer, it is contemplated that themodified cells (i.e. tumor-specific lympocytes) release variousmolecules that adversely affect the tumor by interfering with its bloodsupply, by stimulating the immune system to reject the tumor, or byinterfering with the processes of tumor growth or invasion. Of course,some substances may act upon the immune response as well as upon theblood supply of the tumor, such as IL-1α, HLA or TNF.

There is increasing evidence that a number of cancers are remarkablysensitive to extremely high doses of normally ineffectiveanti-neoplastic drugs. These cancers include malignant melanoma,carcinomas of the stomach, ovary, and breast, small cell carcinoma ofthe lung, and malignant tumors of childhood (including retinoblastomaand testicular carcinoma), as well as certain brain tumors, particularlyglioblastoma.

In another specific embodiment, patients with infections by pathogenicmicroorganisms can be treated with recombinant immortalized cells. Suchrecombinant cells can contain a heterologous gene which is expressed asa product which ameliorates disease symptoms, is toxic to the pathogenwithout significant detriment to the host, or interferes with thepathogen's life cycle, etc. Pathogens which cause infections which maybe treated with recombinant cells according to this embodiment of theinvention include but are not limited to lymphotropic viruses such asHuman Immunodeficiency Virus (HIV, the etiological agent of acquiredimmune deficiency syndrome (AIDS)) (Gallo et al., 1984, Science224:500-503; Barre-Sinoussi, F., et al., 1983, Science 220:868; Levy, J.A., et al., 1984, Science 225:840); gram-negative bacilli such asBrucella or Listeria; the mycobacterium which cause tuberculosis, orwhich cause Hansen's disease (leprosy); parasites such as Plasmodium(the etiological agents of malaria), or Leishmania; and fungi (such asthose that cause pneumonia and other lethal infections secondary toimmunodeficiencies) (for a discussion of many of these disorders, seeHarrison's Principles of Internal Medicine, 1970, 6th Edition, Wintrobe,M. M., et al., eds., McGraw-Hill, New York, pp. 798-1044).

As a particular embodiment, it is possible to construct recombinantcells that express a sequence which is "anti-sense" to the nucleic acidof a pathogen. Such a sequence, which is complementary to the pathogen'sNA or DNA, can hybridize to an inactivate such RNA or DNA, inhibitingthe function or expression of the nucleic acid and disrupting thepathogen's life cycle. As a particular example, recombinant cells can beused in the treatment of AIDS, a disorder which is cased by HIV,apparently by infection of T4 lymphocytes (Dagleish et al., 1984, Nature312:767-768). Recombinant cells which express an antisense nucleic acidthat is complementary to a critical region (e.g., the long-terminalrepeat or polymerase sequence) of the HIV genome (Wain-Hobson et al.,1985, Cell 40:9-17) can be used for the treatment of AIDS.

In a particular interesting aspect of the present invention, lymphocytescapable of producing a specific antibody can be isolated from a patientand immortalized using the process of the present invention. Thenon-tumorigenic immortalized lymphocyte cells can then be injectedrepeatedly into the patient from the immortalized cell line tocontinually supplied antibodies against a target antigen. Suitableantigens include, but are not limited to, AIDS virus antigens such asgp120 and gp41 glycoproteins. Also, immortalization of tumor specificlymphocytes or infectious disease-specific lymphocytes would permit thetreatment of a wide variety of neoplastic and infectious diseases whichinvolve cellular immunity as a protective mechanism.

In addition to cancer, various non-neoplastic diseases may also betreated in accordance with the present invention. For example,hemophilia A and B may be treated with cells expressing Factors VIII andIX. Additionally, thrombotic disorders may be treated with cellsexpressing anti-thrombin III, Protein C or Protein S in patients withdeficiencies of these proteins. Further, endocrine deficiencies may betreated with cells producing growth hormone or insulin. However, abetter gene regulation is required for the insulin gene because itsexpression must be linked to the level of glucose in the blood.

Moreover, bone marrow failure states such as aplastic anemia, cyclicneutropenia, bone marrow hypoplasia following chemotherapy or bonemarrow transplantation may be treated with cells producing G-CSF,GM-CSF, IL-3, M-CSF or c-kit ligand.

Cytokines, such as IL-1α, may be used for modulating an improved immuneresponse to the tumor, and are useful in this dry delivery system.However, inducible promoters may be necessary for IL-1α and TGF-β.

In particular, IL-2 may be used to recruit cytotoxic T lymphocytes (CTL)and natural killer cells directed against tumor cells. Υ-interferon maybe used for induction of class II HLA gene expression by tumor cellsallowing improved recognition of tumor cells by CTLs. Also, TNF-α may beused for augmentation of the immune response to tumor cells and damageto endothelial cells supplying the tumor. It is envisioned that thegenetically-modified endothelial cells will have become incorporatedinto the blood vessels supplying the tumor, and, thus, will be optimallysituated to deliver the TNF in a locally high concentration to thetumor. This should avoid the toxicities that have been observed when TNFis administered systemically. Since the endothelial cells producing theTNF are also susceptible to the effects of the molecule, it will benecessary to place the TNF gene under the control of an induciblepromoter, such as the metallothionein promoter. TNF expression would beturned on only after the endothelial cells have become incorporated intothe blood supply of the tumor. Further, anemia of chronic renal failuremay be treated with cells producing erythropoietin.

Although any donor gene may be used which is capable of expressing aproduct in and secreting it from endothelial cells, mention may be madeof several non-limitative examples.

For example, cells in tumor metastasis deposits may secrete cytokinessuch as IL-1, IL-2, IL-4, TNFα or β, interferon or allogeneichistocompatibility (HLA) antigens to initiate an immune rejection of thetumor, or alternatively secrete agents which would interfere withfurther angiogenesis, such as growth factor receptor-blocking peptides.

The present invention may also be used to effect systemic secretion ofagents, such as Factor VIII or to interfere with the neovascularzationof diabetic retinopathy.

In particular, the following illustrative, but non-limiting examples ofdonor genes may be mentioned: adenosine deaminase (ADA), Factor IX,hematopoietic growth factors (GM-CSF, G-CSF, M-CSF, erythropoietin, kitligand, IL-3), protein C, protein S, hirudin, insulin, growth hormoneand parathyroid hormone.

The transformant cells, regardless of therapeutic utility, aregenetically administered in an amount of about 10⁶ to 10¹² cells/kg ofbody weight. For higher cell numbers, it is preferably to administer thecells by slow infusion.

For example, a cell solution in sterile 5% saline or dextrose-5%-salinesolution may be used having a concentration of about 10⁶ cells/250-500μl. Such a solution can be administered intravenously in about 30seconds. Thus, larger numbers of cells may be conveniently administeredby slow infusion. However, the cell concentration of these solutions maybe varied.

Alternative to infusion, immortalized cells may be directly injectedwith the appropriate target organ. For example, immortalized prostatecells may be directly injected into the prostate to establish foci ofgenetically engineered cells. Approximately 10⁶ /0.25 ml. can beinjected at a single site to establish such immortalized colonies. Therejected cells will first be washed free of growth medium, suspended insterile phosphate buffered saline, and injected with a syringe andneedle. Multiple sites within an organ may be injected depending uponthe necessary requirement for cell number. Additionally, it may benecessary to "wound" the normal epithelium (such as in the case oftrachea or gastrointestinal tract) to permit "seeding" or grafting withthe immortalized epithelial cells.

Subsequent to immortalization by the E6/E7 genes, the established celllines can also be cultured in optimal growth medium to allow for theproduction of either endogenous gene products (such as growth factors,hormones, cell regulatory molecules) which are characteristic of thatparticular cell differentiation state or of exogenous gene productsencoded by DNA transfected into the recipient immortalized host cell.The cellular gene products can consist of either external secretedproteins or internal proteins which are either soluble ormatrix-associated The in-vitro conditions and/or medium optimal cellgrowth have been determined for most human cell types and arecharacteristic for the basic cell types: epithelial and mesenchymal.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES Example 1

Amplification and cloning of the LCR-E6-E7 region of HPV-16 and HPV-18from human tumors.

DNA was extracted and purified from primary human cervical, penile, andvulvar carcinomas. Oligonucleotides corresponding to the 5' end of theLCR (defined as the terminus of the L1 ORF in both HPV-16, bp 7141, andHPV-18, bp 7114) and the 3' end of the E7 ORF (bp 878 for HPV-16 and bp927 for HPV-18) were used to amplify the LCR-E6-E7 region (˜1.6 kb) withinclusive HindIII and Xhol cloning sites as indicated. 100 ng of tumorDNA or 0.1 ng of plasmid DNA were added to a 100-μl reaction mixturecontaining 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01%gelatin, 200 μM each dATP, dTTP, dCTP, and dGTP, 1 μM each primer, and2.5 units of Taq polymerase (Perkin-Elmer Cetus), and submitted to 30cycles of 1 min at 94°, 2 min at 55°, 5 min at 70° in a thermal cycler(Perkin-Elmer Cetus). DNA fragments of the expected size were gelpurified and cloned unidirectionally into a pUC18-derived vectorcontaining the SV40 polyadenylation signals.

Example 2

Generation of Immortalized Human Prostate Cell Line.

The E6/E7 genes of HPV-18 were transfected into primary human prostatecultures (obtained from human fetal tissue, courtesy of Dr. EdwardKaighn) and have been capable of generating immortalized cell lineswhich differ significantly) in their phenotype from those previouslygenerated by SV40 (Kaighn et al., Transformation of human prostateepithelial cells by strontium phosphate transfection with a plasmidcontaining the SV40 early region genes, Cancer Research 49:3050-3056,1989). The primary prostate cells were grown in a serum-free medium(P48F) which contains a mixture of 8 growth factors and were infectedwith a retrovirus containing the HPV-18 LCR/E6/E7 region. The cells werepassaged (1:2 split) until immortalized clones were detected. The cellswere cuboidal, epithelial cells and differed significantly from the morefibroblastic appearance of the SV40-immortalized prostate cells.

Example 3

Cystic Fibrosis Generation of Immortalized Human Tracheal Cell LinesFrom Cystic Fibrosis Patients.

Primary culture.

Donor tissue was obtained postmortem from a 24 year old man with cysticfibrosis who was homozygous for the phenylalanine 508 deletion in theCystic Fibrosis Transmembrane Conductance Regulator (CFFR) gene. Thetrachea was cut into 2×2 cm pieces and washed with Joklik's modifiedessential medium (MEM) containing antibiotics, dithiothreitol (0.5mg/ml), and DNAse 10 μg/ml) at 4 degrees C. for 3 hours. The tissueswere then incubated in fresh supplemented MEM plus protease (Sigma TypeXIV, 0.1 μg/ml) at 4 degrees for 18 hours. The epithelial cells weredislodged by gentle agitation and plated in hormone-supplemented F12medium (FI2+7x; supplements:insulin 5 μg/ml, endothelial cell growthsupplement 3.7 μg/ml, epidermal growth factor 25 ng/ml, triiodothyronine3×10-8M, hydrocortisone 1×10-6M, transferrin 5 μg/ml, and cholera toxin10 ng/ml, plus ceftazidime, tobramycin, amphotericin B).

Transfection with HPV-18 E6 and E7 genes.

A pUC19-based plasmid containing the HPV-18 nucleotides 6273-2440encoding the intact E6 and E7 open reading frames, a partial E1 openreading frame, and the upstream regulatory region was transfected bylipofection as described. After a 2 hr incubation at 37, 12 ml of freshF12+7x medium was added. On the following day the cells were fed withfresh medium.

Culture and Clonal selection.

At 14-18 days post-seeding, clusters of 30-200 dividing cells ofapparent clonal origin developed and were isolated using cloningcylinders. Between passages 1-4, most subclones were co-cultured withlethally irradiated NIH3T3 fibroblasts, which were removed bydifferential trypsinization at passage 4. Eleven clones were isolatedand developed a polygonal morphology typical of air-way epithelial cellsin primary culture.

Presence and expression of HPV genes in immortalized cell lines.

The presence of the HPV-18 genome in selected clones was assayed usingpolymerase chain reaction (PCR) technology with oligonucleotide primersspecific for the HPV-18 E6-E7 region. The 5' primer corresponds toHPV-18 nucleotides 105-124 and the 3' primer to nucleotides 888-907 ofthe HPV-18 DNA sequence. Extracts of 6×10³ cells of selected clones wereanalyzed by PCR for 30 cycles with the following conditions: 94 C. for 1min, 50 C. for 2 min, and 72 C. for 3 min. An HPV-18 transformed humankeratinocyte cell line (18Nco) and an SV40-transformed keratinocyte cellline were used as positive and negative controls. Agarose gelelectrophoresis of PCR products demonstrated the 802 bp E6-E7 amplifiedproduct in the positive control and in all CF clones examined.

Expression of the HPV-18 E7 protein.

Ten cm dishes of selected clones were metabolically labelled with ³⁵S-cysteine for 4 hours following a 2 hr starvation in cysteine-freemedia. Total protein was extracted following labelling andimmunoprecipitated with 20 μl of a rabbit polyclonal antibody specificfor the HPV-18 E7 protein. The immunoprecipitated proteins wereseparated electrophoretically on a 14% acrylamide-SDS gel.Autoradiography of the gel showed the presence of the 17 kD E7 proteinin both of the CF clones examined as well as the 18-Nco positive controland absent from the SV40 negative control. A combinedimmunoprecipitation/immunoblotting procedure was also used to detect theE7 protein. Cell extracts were immunoprecipitated as above andelectrophoretically separated. The gel was then blotted ontonitrocellulose and the E7 protein was detected by Western blotting usinga Protoblot (Promega) kit using a 1:100 dilution of the rabbitpolyclonal antibody as primary antibody. The 17 kD E7 protein wasdetected in all clones examined.

Ion Transport Properties.

To screen for the development of functional tight junctions, clones werepassaged onto a collagen matrix support. Beginning on day followingpassage, transepithelial resistance (Rt) and spontaneous transepithelialpotential difference (Vt) were measured daily using a WPI electrometerconnected to the apical and basolateral media with calomel half-cells.Measurements were taken daily until the Vt declined or the cellssenesced. Resistance was calculated from the voltage deflections inducedby ±7 μamp current pulses passed through silver- silver chlorideelectrodes placed in the mucosal and submucosal bathing solutions.Transepithelial resistance (RE) for the CF lines (CFTI) is similar tothat observed in primary cultures of human airway epithelial cells,indicating the presence of tight junctions, while R_(t) forSV40-transformed cells (CF/T43) is markedly decreased. Additionally, thetransepithelial potential difference (V_(t)) in CF lines is -13.3 whichis approximately 5-fold higher than V_(t) in SV40-transformed airwayepithelial cells.

Heterologous tracheal grafts.

To assess the morphologic differentiation potential of CFT1 cells,heterologous rat tracheal grafts were prepared as described. In brief,the native epithelium was removed from excised tracheas of Fisher 344male and female rats by freezing in liquid nitrogen, thawing at roomtemperature, and flushing the lumen with phosphate-buffered saline. Thefreeze-thaw cycle was repeated twice. A suspension of 6×10⁵ CFT1 cells(passage 13) in 50 μl F12+7X or CF/T43 cells (passage 16) was injectedin the lumen of each trachea, the ends were closed with sterile 2-0 silksuture, and the tracheas were grafted in the subcutaneous space of C57BLnude mice. After 1 to 6 wk, the grafts were removed, opened, and fixedin freshly-prepared 4% paraformaldehyde. After standard dehydration andparaffin embedding, 5 micron sections were cut and stained withRichardson's, hematoxylin and eosin (H & E) or Alcian blue-period acidSchiff (AB-PAS) stains.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGTATTAAGCTTAACGTAAGCTGTAAGTATTG32                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTATCAAAGCTTTGCGTGTACGTGCCAGGAAG32                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGTATTAAGCTTTGTATGATTGCATTGTATGG32                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGATGGTACCGACTAGGACGGAGCTCCGATTA32                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGTTGTTACCGACTAGGTCTGAGCTCAGTAGC32                                            __________________________________________________________________________

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A non-tumorigenic immortalized human epithelialcell line which retains the phenotypic properties of the parentepithelial cells used for immortalization which is produced by a methodcomprising;(i) transfecting epithelial cells selected from the groupconsisting of epithelial cells lining the oral and nasal mucosa, larynx,trachea, lung, esophagus, stomach, duodenum, jejunum, ileum, colon,liver, pancreas, kidney, bladder, adrenal, hair follicle and dermalpapillae epithelial cells with a DNA vector containing a subgenomicfragment of a human papillomavirus type 16 or 18 comprising the E6 andE7 genes or the E7 gene of human papillomavirus type 16 or 18; and (ii)selecting for an immortalized non-tumorigenic epithelial cell line whichpossesses the phenotypic properties of the parent epithelial cells. 2.The immortalized epithelial cell line of claim 1 wherein said cell lineis a respiratory epithelial cell line.
 3. The immortalized epithelialcell line of claim 1 wherein said cell line is a tracheal epithelialcell line.
 4. The immortalized epithelial cell line of claim 3 whereinsaid human tracheal epithelial cell line is a human cystic fibrosistracheal epithelial cell line.
 5. The immortalized cell line of claim 1wherein the DNA vector additionally contains a heterologous DNA sequenceencoding for a desired protein or polypeptide.
 6. The immortalizedtracheal epithelial cell line of claim 3, wherein said cell line retainsthe ion transport properties of the parent epithelial cells.
 7. Theimmortalized human cystic fibrosis tracheal epithelial cell line ofclaim 4, wherein said cell line retains the ion transport properties ofthe parent human cystic fibrosis tracheal epithelial cell line.